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Point of Care Biosensor System for Nucleic Acid Based Ion-Mediated Real-Time Analysis of Hepatitis C Virus

Thesis Info

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Author

Naqeeb Ullah

Program

PhD

Institute

International Islamic University

City

Islamabad

Province

Islamabad.

Country

Pakistan

Thesis Completing Year

2018

Thesis Completion Status

Completed

Subject

Electrical Engineering

Language

English

Link

http://prr.hec.gov.pk/jspui/bitstream/123456789/11064/1/Naqeeb%20Ullah_Biotech_2018_IIU_PRR.pdf

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676727811144

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Hepatitis C virus (HCV) is one the major threat to public health being leading cause of chronic liver disease with 92 to 149 million people affected worldwide. A rapid and accurate diagnosis is the key to prevent viral transmission and management of disease progression. A point-of-care (POC) system was designed for rapid detection of HCV infection called as STALLION (Single Tube Analysis using LAMP, LED and ION-sensing).Viral RNA was initially isolated using conventional spin-column and magnetic bead based methods, however, for POC setup, two modified methods were described. The first method uses a millifluidic channel with nucleic-acid-binding-silica-membrane made with the help of a novel approach called grid sandwich method (GSM).This method uses plastic canvas grid to make fluidic channels and then canvas grid is sandwiched between two sheets of polymeth-methacrylate (PMMA) sheets and fixed with the help of suitable adhesive. The second method employs transparent soft tube with reagents separated by surface tension valves and isolation is performed by nucleic-acid-bindingmagnetic-beads. The later approach was used with the currently designed STALLION system for isolation of RNA. A custom detection probe was made to dip into PCR tube comprising an ion sensitive field effect transistor (ISFET), a micro-capillary based Ag/AgCl reference electrode and a temperature sensor. In-vitro replication of HCV genomic RNA was performed by reverse transcription loop-mediated isothermal amplification (RT-LAMP) and detected in real-time by ISFET bio-sensing of released H+ ions. Heat incubation at 60°C was provided by a very low cost novel concept using dissipated heat from power LED. With this system, the HCV positive samples with 101, 102, 103 copies/ml were detected in as less as 30, 18 and 13 minutes respectively against no-template control (NTC). Detection limit was comparable to available methods such as nested PCR. No significant change was observed for HCV negative samples.The results can be viewed on associated LCD screen or alternatively can be transferred to computer or smart phone. STALLION system provides analysis in conventional PCR tube format avoiding any complex fluidics or instrumentation requirements. Such a system will be particularly useful for POC, rapid and reliable clinical diagnosis of HCV RNA with no prior expertise in limited resource settings especially for developing countries.Further, STALLION system was tested for detection Malarial parasite which is a DNA containing infectious agent. The testing was also performed using fluorescent dye and custom designed real-time detection setup. These experiments showed that STALLION system can be effectively used for the detection of any organism whose nucleic acid can be amplified with LAMP in POC settings making it suitable and convenient for testing especially in remote and under-developed areas. In conclusion, the STALLION setup provides a complete system to perform RT-LAMP (from RNA source) or LAMP (from DNA source), and easy scalability for performing other isothermal nucleic acid amplification tests using specific primer sets and standardization accordingly.
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85. Al-Buruj/The Constellations

85. Al-Buruj/The Constellations

I/We begin by the Blessed Name of Allah

The Immensely Merciful to all, The Infinitely Compassionate to everyone.

85:01
a. By the celestial realm full of constellations,

85:02
a. and the Promised Time,

85:03
a. and a witness and a witnessed.

85:04
a. Destroyed were the People of the Ditch,

85:05
a. of the fire abounding in fuel, it will be fueled,

85:06
a. as they gathered around it,

85:07
a. witnessing to what they were doing to the believers,

85:08
a. and they avenged them merely because they believed in Allah – The One and Only God,
b. The Almighty, All-Praiseworthy,

85:09
a. Allah - the One WHO holds the reign over the celestial realm and the terrestrial world,
b. and Allah is Witness to everything.

85:10
a. Surely, for those who oppress and persecute the believing males and the believing females,
b. and then do not repent of their evil,
c. for them will be severe suffering of Hell,
d. added to that shall also be suffering of the Blazing Fire for having tortured to death the believers.

726 Surah 85 * Al-Buruj

85:11
a. Indeed, those who believe and practice righteousness, for them will be Paradise under which rivers/streams flow.
b. Such will be the great success!

85:12
a. Nevertheless, the grip/onslaught of your Rabb - The Lord against the disbelievers will be intense and painful.

85:13
a. It is, indeed, HE WHO initiated, and
b. will repeat it when HE will choose to.

سوشل میڈیا اور اسلامی جمہوریہ پاکستان : ثقافتی مبارزہ،چیلنجزاور ممکنہ حل Social Media and the Islamic Republic of Pakistan: Clash of Civilizations, Challenges and Possible Solutions

This research explores the dynamic interaction between social media and the Islamic Republic of Pakistan, revealing a complex culture war unfolding in the digital landscape. The study examines how social media platforms have become arenas of competition, where traditional Islamic values collide with the global influence of digital communication. By examining the ways in which social media have shaped and reflected Pakistani cultural identity, the research examines the tension between modernity and conservatism, as well as challenges to established social norms. Additionally, it analyzes the role of social media in promoting communication, political discourse, and diverse voices in the context of Pakistan's cultural landscape. By shedding light on this culture war, this research contributes to understanding the complex relationship between social media and the Islamic Republic of Pakistan. Keywords: Social media, Islamic Republic of Pakistan, Clash of Civilizations, digital communication, Islamic Culture.

Characterization of Chilli Anthracnose Pathogen and its in Vitro Management

Chilli anthracnose is the most prevalent disease in all major chilli growing areas of the Punjab. Chilli anthracnose is causing the highest yield losses than any other fungal disease. This study was focused to assess the prevalence and incidence of the disease in five major chilli production districts of Punjab. Moreover, morphological and molecular characterization of pathogen and invtiro management by using different techniques of application through five plant extract. Field based surveys were conducted in two years (2014-2015 and 2015-2016) for disease documentation and sample collection followed bylaboratory isolations resulted into the recovery of 142 pathogenic isolates of the pathogen associated with the disease. Disease was recorded in all surveyed area and showed 100% prevalence viz., Vehari, Bahawal Nagar, Okara, Kasur and Rawalpindi. The maximum average incidence 63.10% was recorded at Vehari while minimum average incidence 25.5% was recorded at Rawalpindi.Morphological characterization of the recovered isolates showed variation in morphological characters like two types of colony colours were observed. Fusiform and falcate conidial shapes were recordedand some isolates produced setae while other not. Variations in number of acervuli were also observed. Molecular characterization was done including DNA sequencing of pathogenic isolates though PCR amplification of the Internal Transcription Spacer region using ITS1 and ITS4 primers. Characterization established the identity of the highly pathogenic isolates selected from each district to their morphological characterization done before molecular study. Phylogenetic analysis of 5 morphologically and pathogenically diverse Colletotrichum capsici xxii isolatesexpressed their evolutionary relationship by 99-100% similarity with previously reported isolates available at NCBI database. The most virulent isolate CCO41was used as source of inoculum in in vitro management of the pathogen by using five different plant extracts applied through two different techniques viz., poisoned food technique and well diffusion technique. All applied plant extracts showed antifungal properties and maximum 98.5% growth inhibition was noted down in case of polygonum plant extract @ 0.1% and 0.2% when applied through poisoned food technique while minimum antifungal activity was expressed by dodonaea and clove at the dose of 25μL when applied through well diffusion technique. Poisoned food was found better than well diffusion technique in the study. Results of the study generated exact and comprehensive picture of the disease in major chilli growing areas of the Punjab and indicated the areas at high risk in terms of disease spread. The survey observations also pointed out the trend of decreasing chilli crop area in the all districts due to number of factors including anthracnose. Isolates recovered from the study area were first time studied with details with reference to morphological and molecular characteristics.In vitrobiologicalmanagement studies explored the antifungal potential of indigenous plant against Colletotrichum capsici. Polygonum plant extract was found to be the most effective by suppressing pathogen’s growth up to 98.5% and depicted its antifungal potential.These management findings indicate projected antifungal ability of plant extracts to manage chilli anthracnose in field conditions.